Cervical cancer (CC) remains a significant global health burden, particularly in low- and middle-income countries, where it is a leading cause of cancer-related mortality among women. Annually, more than 600,000 new cervical cancer cases are diagnosed worldwide, with a 53% mortality rate. The development of CC is strongly associated with a persistent infection with high-risk human papillomavirus (HR-HPV), particularly HPV16 and HPV18, which are responsible for 70% of cases. This persistent HPV infection leads to uncontrolled proliferation of infected cells via downregulation of epithelial differentiation, disruption of epidermal development, and evasion of innate immune responses. The oncogenic potential of HR-HPV types is primarily attributed to the expression of E6 and E7 oncoproteins, which are integral to cancer phenotype maintenance in HPV-infected cells. Despite the implementation of preventive strategies, such as prophylactic vaccination and screening, which have contributed to a reduction in disease incidence, there remains an urgent need for targeted therapeutic interventions aimed at addressing persistent HPV infection and advanced cervical lesions. The HR-HPV E6 protein is composed of 158 amino acids and 2 zinc-binding motifs, and its most important function is escaping cell death by the induction of p53 degradation. In fact, E6 interacts with the LxxLL motif of the E6-associated protein (E6AP), an E3 ubiquitin ligase, inducing p53 degradation through the proteasome pathway and, consequently, blocking p53-dependent apoptosis. The HR-HPV E7 protein has around 98 amino acids, with an N-terminal region intrinsically disordered, whereas the C-terminal features a well-structured zinc-binding site. One of the most important protein regulation mechanisms by the E7 protein is through the retinoblastoma (pRb)/E2F system. The E7 protein binds to the pRb through the motif LxCxE, displacing E2F complexes, allowing its release and, as a result, the transcription of S-phase genes. This ensures that the infected cells remain in an S-phase-competent state, leading to hyperproliferation. Considering their crucial role in driving cellular transformation and cancer maintenance, both proteins are primary targets for developing targeted HPV therapies. However, the rational design and optimization of therapeutic strategies may depend on the ability to obtain both proteins. Unfortunatelyss, isolating E6 and E7 proteins from human cells is not feasible, so recombinant technology is a great alternative. Nonetheless, challenges persist related to the low solubility and tendency of these proteins to aggregate. Overcoming these issues is essential for enabling structural studies, biophysical characterization, and high-throughput screening of potential inhibitors. Thus, this thesis proposes the implementation of a biotechnology platform, starting with the biosynthesis towards the purification of the HPV16 E6 and E7 proteins, to ultimately discover inhibitory drugs that will be able to block the oncogenic effects triggered by both targets through biophysical and in vitro approaches. Firstly, we established a biotechnological platform for the recombinant expression and purification of HPV16 E6 in Escherichia coli BL21, employing a dual-tagged construct to enhance yield and address solubility issues. A two-step chromatographic process: affinity chromatography, followed by immobilized-metal affinity or size exclusion chromatography, obtained highly pure protein fractions (~98%). Circular dichroism confirmed that the secondary structure of His6-MBP-E6 protein is preserved in both purification strategies. Additionally, thermal shift assay (TSA) suggested potential additives for protein stabilization, such as maltose, glycerol, and detergents that can be incorporated in further characterization studies. In parallel to obtain the E6 protein, we focused on identifying natural compounds that may disrupt the E6/E6AP interaction using virtual screening. Three candidates selected from in silico studies with favorable predicted binding affinities were evaluated in HPV-negative and HPV-positive cell lines. Taxifolin and lucidin protected p53 from E6-mediated degradation and reactivated its transcriptional activity, leading to apoptosis induction. Nevertheless, neither compound affected cell cycle progression; thus, we extended the search for E6 inhibitors targeting directly the p53 binding site by exploring four distinct families of small molecules (barbiturates, dihydropyrimidinones/thiones, acetanilides and steroid derivatives). The screening was achieved through a combination of computational strategies and biophysical assays using the recombinant E6 protein previously produced. Fluorescence-based studies suggested that 4 compounds displayed a moderate affinity for the E6 protein. These candidates (CMP44, CMP50, CMP10c and CMP11c) were further characterized in HPV-positive and HPV-negative cell lines. The results demonstrated that CMP50 and CMP11c effectively rescued p53 from E6 degradation, reducing the proliferation and migration of HPV-positive cancer cells. Additionally, both compounds reactivated p53-dependent transcriptional factors, activating an apoptotic pathway and inducing cell cycle arrest, which suggests their therapeutic potential against HPV. Finally, considering the aim is also to study the E7 protein and potential inhibitors, we explored and characterized the dual-tagged recombinant HPV16 E7 protein. Following a similar workflow to what we had built for the E6 protein, we explored several chromatographic approaches, including affinity, anion-exchange, mixed-mode, and sizeexclusion chromatography, to obtain the His6-MBP-E7 protein. A thorough analysis of buffer compositions and additives indicated that a pH between 6.0 and 7.0, as well as sugars and detergents, maximized E7 solubility and minimized sample heterogeneity. In this process, we obtained the target protein with high yield, a purity of around 98% as confirmed by LC-MS/MS analysis. Additionally, the inclusion of detergents in the final formulations was studied, where the inclusion of DM (η-Decyl-β-D-Maltopyranoside) or η-Dodecyl-β-D-Maltopyranoside (DDM) led to a monodisperse sample. The optimized protocol is suitable for downstream structural and functional characterization, as well as for future inhibitor screening studies. Collectively, the work presented in this thesis establishes a reproducible platform for the production and study of the HPV16 E6 and E7 oncoproteins, which can be used for expressing proteins from other HPV types. Additionally, by integrating computational, biophysical, and cellular approaches, this research identified specific anti-HPV drugs, which can be the starting point for structural studies aiming at obtaining structures of the protein-inhibitor complexes.

Gellan gum, a microbial exopolysaccharide, is biodegradable and has potential to fill several key roles in many fields from food to pharmacy, biomedicine and tissue engineering. In order to improve the physicochemical and biological properties of gellan gum, some researchers take advantage of numerous hydroxyl groups and the free carboxyl present in each repeating unit. As a result, design and development of gellan-based materials have advanced significantly. The goal of this review is to provide a summary of the most recent, high-quality research trends that have used gellan gum as a polymeric component in the design of numerous cutting-edge materials with applications in various fields.

This work demonstrates the potential of calcium- and nickel-crosslinked Gellan Gum (GG) microspheres to capture the Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) directly from complex Komagataella pastoris mini-bioreactor lysates in a batch method. Calcium-crosslinked microspheres were applied in an ionic exchange strategy, by manipulation of pH and ionic strength, whereas nickel-crosslinked microspheres were applied in an affinity strategy, mirroring a standard immobilized metal affinity chromatography. Both formulations presented small diameters, with appreciable crosslinker content, but calcium-crosslinked microspheres were far smoother. The most promising results were obtained for the ionic strategy, wherein calcium-crosslinked GG microspheres were able to completely bind 0.1% (v/v) DM solubilized STEAP1 in lysate samples (~7 mg/mL). The target protein was eluted in a complexed state at pH 11 with 500 mM NaCl in 10 mM Tris buffer, in a single step with minimal losses. Coupling the batch clarified sample with a co-immunoprecipitation polishing step yields a sample of monomeric STEAP1 with a high degree of purity. For the first time, we demonstrate the potential of a gellan batch method to function as a clarification and primary capture method towards STEAP1, a membrane protein, simplifying and reducing the costs of standard purification workflows.

Quercetin is a natural flavonoid with high anticancer activity, especially for related-HPV cancers such as cervical cancer. However, quercetin exhibits a reduced aqueous solubility and stability, resulting in a low bioavailability that limits its therapeutic use. In this study, chitosan/sulfonyl-ether-β-cyclodextrin (SBE-β-CD)-conjugated delivery systems have been explored in order to increase quercetin loading capacity, carriage, solubility and consequently bioavailability in cervical cancer cells. SBE-β-CD/quercetin inclusion complexes were tested as well as chitosan/SBE-β-CD/quercetin-conjugated delivery systems, using two types of chitosan differing in molecular weight. Regarding characterization studies, HMW chitosan/SBE-β-CD/quercetin formulations have demonstrated the best results, which are obtaining nanoparticle sizes of 272.07 ± 2.87 nm, a polydispersity index (PdI) of 0.287 ± 0.011, a zeta potential of +38.0 ± 1.34 mV and an encapsulation efficiency of approximately 99.9%. In vitro release studies were also performed for 5 kDa chitosan formulations, indicating a quercetin release of 9.6% and 57.53% at pH 7.4 and 5.8, respectively. IC50 values on HeLa cells indicated an increased cytotoxic effect with HMW chitosan/SBE-β-CD/quercetin delivery systems (43.55 μM), suggesting a remarkable improvement of quercetin bioavailability.

Cervical cancer (CC) is the fourth most common pathology in women worldwide and presents a high impact in developing countries due to limited financial resources as well as difficulties in monitoring and access to health services. Human papillomavirus (HPV) is the leading cause of CC, and despite the approval of prophylactic vaccines, there is no effective treatment for patients with pre-existing infections or HPV-induced carcinomas. High-risk (HR) HPV E6 and E7 oncoproteins are considered biomarkers in CC progression. Since the E6 structure was resolved, it has been one of the most studied targets to develop novel and specific therapeutics to treat/manage CC. Therefore, several small molecules (plant-derived or synthetic compounds) have been reported as blockers/inhibitors of E6 oncoprotein action, and computational-aided methods have been of high relevance in their discovery and development. In silico approaches have become a powerful tool for reducing the time and cost of the drug development process. Thus, this review will depict small molecules that are already being explored as HR HPV E6 protein blockers and in silico approaches to the design of novel therapeutics for managing CC. Besides, future perspectives in CC therapy will be briefly discussed.